Understanding the isomerization of the HIV-1 dimerization initiation domain by the nucleocapsid protein

Abstract

The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC-hinge interactions were observed in constructs including at least one unpaired guanine at the 5'-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3'-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5'-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.

Figure 1

Nanospray-FTICR mass spectra of samples containing 30 μM NC (●) and 10 μM of either a) wild-type SL1B (◆), or b) SL1B-A272G (▽) in 150 mM ammonium acetate (pH 7.0) at room temperature. The signal corresponding to the −7 ion of the NC-SL1B complex is enlarged in the inset to show how the charge state of each species is unambiguously determined from the respective isotopic spacing. The corresponding molecular mass is then readily obtained from the charge state and the position of the peak on the m/z scale. Note that a maximum 2:2 protein to RNA ratio was observed for the KL assembly of wild-type SL1B, whereas a single base substitution in the junction region allowed for the binding of a third equivalent of NC.

Figure 2

Product ion spectra obtained by submitting a) the KL-obligated dimer (■□) and b) the ED-obligated dimer (◀▷) to mild SORI-CID (see Materials and Methods). Panel c) was obtained by submitting the ED-obligated dimer to harsher activation conditions. Boxed symbols identify each precursor ion. Note that the KL dimer readily dissociated into its monomeric components, whereas the ED conformer remained intact under identical activation conditions. At more energetic regimes, the latter provided typical d-H₂O and y series, which were produced by covalent fragmentation of the RNA backbone.

Figure 3

Product ion spectra obtained by submitting the a) 1:2, b) 3:2, and c) 4:2 complexes of NC (●) and ED-obligated dimer (◀▷) to mild SORI-CID (see Materials and Methods). Boxed symbols identify each precursor ion. Note that the sequential loss of NC units constitutes a common thread among these spectra, whereas no dimer dissociation was observed. The fact that the 2:2 product was the predominant species obtained from the higher order precursors provides a measure of the intrinsic kinetic stability of this assembly.

Figure 4

Product ion spectra obtained by submitting the a) 1:2, b) 2:2, and c) 3:2 complexes of NC (●) and KL-obligated dimer (▲△) to mild SORI-CID (see Materials and Methods). Boxed symbols identify each precursor ion. Note that both NC loss and dimer dissociation were observed in these spectra.

Figure 5

a) Nanospray-FTICR mass spectrum of a sample containing 30 μM NC (●) and 10 μM wild-type SL1A (×) in 150 mM ammonium acetate (pH 7.0) at room temperature. Panel b) and c) include the product ion spectra obtained by submitting the 1:2 and 3:2 complexes to mild SORI-CID (see Materials and Methods). Boxed symbols identify each precursor ion. Note that the 3:2 complex represented the maximum stoichiometry observed in this experiment and that activation of the different precursor ions resulted in both NC loss and dimer dissociation.

Figure 6

a) Nanospray-FTICR mass spectrum of a sample containing 30 μM NC (●) and 10 μM wild-type SL1A (×) in 150 mM ammonium acetate (pH 7.0) after 3 hour incubation at 37°C. Panel b) and c) include the product ion spectra obtained by submitting the 3:2 and 4:2 complexes to mild SORI-CID (see Materials and Methods). Boxed symbols identify each precursor ion. In panel b) and c), the percentages in parentheses indicate the normalized intensity of the corresponding signal compared to the total intensity of the product species, as described in Materials and Methods. Dimeric products were assigned to the ED conformer, whereas monomeric products were assigned to KL. The percentages were then summed to determine the partitioning between the two conformers within each precursor ion population, as reported in the box. In panel a), the KL/ED proportions within each ion signal were used to obtain the overall proportions of monomeric, KL, and ED forms in solution (summarized in Table 2).

Figure 7

a) Loop-loop interactions in the KL dimer formed by wild type SL1A (PDB: 1XPF). The junction bases are labeled according to the subtype A sequence (Mal variant). The arrows give an idea of the putative rearrangements that may take place during the isomerization process. The graphical rendition in panel b) was created using Pymol to visualize the expected stacking between hinge and loop bases to extend the initial loop-loop helix. The arrows show the possible unzipping of the intramolecular base pairs of the KL dimer, which is necessary to enable strand exchange. The graphical rendition in panel c) depicts a more advanced stage of strand exchange, in which a greater number of intermolecular base pairs are being formed in concerted fashion. The final result of the isomerization process consists of a full-fledged duplex structure d), represented here by the palindromic region of the ED dimer of wild type SL1A (PDB: 1Y99).

Scheme 1

Two-step model of SL1 dimerization and isomerization. KL stands for kissing loop and ED for extended duplex. The sequence shown here corresponds to the subtype B (Lai variant) of HIV-1, but the nucleotides are numbered according to the subtype A sequence (Mal variant) for the sake of consistency. Stem- and flanking-bulges are highlighted in gray.

Scheme 2

Sequences and secondary structures of the constructs included in the study. Nucleotides are numbered according to the subtype A sequence (Mal variant) of HIV-1. The self-complementary sequences are highlighted in gray. Stems and flanking bulges of dimeric species are also highlighted in gray. For each species, the monoisotopic masses observed experimentally and calculated from sequence are included.